Extended Data Figure 5: Effect of cone- and Rb-related circuitry on cone precursor response to Rb depletion. | Nature

Extended Data Figure 5: Effect of cone- and Rb-related circuitry on cone precursor response to Rb depletion.

From: Rb suppresses human cone-precursor-derived retinoblastoma tumours

Extended Data Figure 5

A, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cells (a), among RXRγ+, CRX+ cells (b), or among cone arrestin+, CRX+cells (c); and percentage of L/M-opsin+, CRX+ cells among all cells with DAPI+ nuclei (d) after transduction of dissociated FW18 retina with shRB1-733 and shRNAs against p130, p107, TRβ2, SKP2, MDM2 and MYCN. B, Percentage of Ki67+ cells among L/M-opsin+, CRX+ cone-like cells (top) and proliferative response (bottom) after transduction of dissociated FW18 retina with shRB1-733 and with shRNAs against RXRγ and p27 (shRNAs 856+930), or with overexpression of p27 and p27-T187A. C, High-level Thr 187 phosphorylated p27 (p-p27(T187), top) coinciding with downregulation of total p27 (bottom) and prominent Rb during cone precursor maturation. C, a, Perifoveal region of FW18 retina. C, b, Enlarged view of boxed regions in C, a. Arrows, cone precursors identified by large, strongly Rb+ nuclei and lack of p27 signal in characteristic outer nuclear layer position7,16. D, Effect of two RBL1-p107 or two RBL2-p130 shRNAs on proliferation of Rb-depleted isolated cone precursors. E, Knockdown efficacy of two RBL1-p107 or two RBL2-p130 shRNAs in Y79 and RB177 retinoblastoma cells. F, Impaired proliferation of Weri-RB1 retinoblastoma cells after transduction with BN-p130 compared to vector control. G, Impaired proliferation of RB177 retinoblastoma cells following transduction with two p107 shRNAs. H, I, Impaired proliferation and MYCN expression in Y79 cells after p107 knockdown with two p107-directed shRNAs, and rescue by shRNA-resistant BN-p107 constructs. J, p27 accumulation and growth suppression following p107 knockdown with shp107-2 rescued by BN-p107-2r in RB1 wild type SKN-BE(2) neuroblastoma cells. p107 overexpression impaired SKN-BE(2) growth, contrary to its effects in Y79. *P < 0.05, **P < 0.01 (compared to SCR or vector control); †P < 0.05, ††P < 0.01 (compared to RB1-KD plus SCR or RB1-KD plus BN vector); ‡P < 0.05, ‡‡P < 0.01 (compared to shp107-2 plus BN vector) (HJ). Data are representative of more than two independent experiments except for SKN-BE(2) analyses. Values and error bars are mean and s.d. of triplicate assays.

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